Stem cell (SC) is a type of pluripotent cells of self-replicating ability which can differentiate into various types of functional cells. Nowadays, mesenchymal stem cell is one type of adult stem cells, which has pluripotent self-replicating ability and has attracted much attention. Mesenchymal stem cell can be cultured and amplified in vitro, and can differentiate into several tissue cells such as bone cells, cartilage cells, fat cells, nerve cells, muscle cells and so on.
In recent years, bone marrow stem cells, umbilical cord stem cells and adipose-derived stem cells (ADSCs) are studied intensively. Adipose-derived stem cell is one type of stem cells having pluripotent self-replicating ability and isolated from adipose tissue. ADSCs have several advantages over bone marrow stem cells and umbilical cord stem cells. It has been found in the research that ADSCs can proliferate in vitro steadily with low decline rate, while it is easy to obtain sources for ADSCs and a large amount of ADSCs can be obtained from a small amount of tissue. ADSCs are appropriate for large-scale culture and grow homogenously while damage to body is small. Moreover, ADSCs have wide resources and large amount of storage in body and ADSCs are appropriate for autoplastic transplantation, and highly safe. The establishment of adipose-derived stem cell library has become a new focus in recent years.
Since the isolation and culturing of human adipose-derived mesenchymal stem cells need a long period, it takes about two weeks or more for primary cells to grow to confluence, and about three weeks to reach certain amount of cells. Long term in vitro cultivation and passage of human adipose-derived mesenchymal stem cells usually leads to a spontaneous differentiation, which results in losing of multi-differentiation potential. Therefore, in order to ensure the continuity of the experiment, it is necessary to provide seed cells in large amounts for experiments or tissue engineering at any time. Therefore, it is necessary to establish an effective method of cryopreservation. The reliability of cryopreservation methods is identified by detecting cell cycle, cell phenotype and adipogenic differentiation potential of human adipose-derived mesenchymal stem cells after cryopreservation and recovery.
At present, the cryopreservation usually adopts serum for cryopreservation. Serum is the greatest used natural medium in cell culture, and its main advantages are as follows:
(1) It contains plenty of nutrients necessary for cell growth so as to sufficiently support cell growth.
(2) The plenty of serum proteins play a protective role on cells.
(3) Growth factors and trace elements essential for cell proliferation are provided for in vitro cell culture;
(4) It provides adherence factors for adherence-dependent cells.
However, there are various disadvantages in using animal serum in freezing medium, mainly including:
(1) Serum contains substances which are toxic for cells, such as polyamine oxidase which can react with polyamines (e.g., spermine, spermidine) from highly reproductive cells, thus forming polyspermine which is cytotoxic. Complement, antibodies and bacteria toxins affect cell growth, and even cause cell death;
(2) Depending on individual animal, origin of serum, and batch number of serum, the quality of each batch of serum varies greatly and it is possible to maintain the consistency of composition.
(3) During the collecting of materials, mycoplasma and viruses may be bought into the serum and cause potential effects on cells, resulting in failure of experiments or unreliable experimental results.
(4) The cost of serum is quite expensive and up to 3000-4000 RMB per 500 ml of serum.
(5) Acquisition of serum from a living body is harmful to animal.
So far, there is no serum-free freezing medium which strictly suitable for adipose-derived stem cells or establishment of adipose-derived stem cell library. Therefore, it is urgent need in the art to develop a new and effective serum-free freezing medium suitable for adipose-derived stem cells.